This invention relates to modified xcex1-bungarotoxin molecules and to methods for selectively inhibiting neurotransmitter receptors using modified and unmodified xcex1-bungarotoxin molecules. More specifically, the invention relates to the use of xcex1-bungarotoxin compositions for the localized inhibition of neuronal and/or muscle cell function.
At the neuromuscular junction, the nicotinic acetyicholine receptor (nAChR) mediates muscle contraction through the binding at the muscle membrane surface of acetyicholine, which is released from the skeletal motorneuron. There is growing evidence that nAChRs in the nervous system play critical roles in a wide variety of physiological responses and pathological states (Sargent, Annu. Rev. Neurosci. 16:403-443, 1993; McGehee and Role, Annu. Rev. Physiol. 57:521-546, 1995; Boyd, Crit. Rev. Toxicol. 27:299-318, 1997), and mediate the behavioral effects of nicotine (Dani and Ileinemann, Neuron 16:905-908, 1996). The class of snake venom proteins known as a-neurotoxins, which are competitive antagonists of nAChRs, have been extremely useful tools in the study of the al-subunit containing muscle-type nAChR and the xcex17-subunit containing neuronal nAChR (Endo and Tamiya, in Snake Toxins A.L. Harvey, Ed., pp 165-222, Pergamon Press, Inc., New York, N.Y., 1991; Chiappinelli, in Natural and Synthetic Neurotoxins, A. Harvey, ed., pp.65-128, Academic Press, New York, N.Y., 1995). xcex1-Neurotoxins have a highly conserved fold, due primarily to four invariant disulfide bonds and are classified as either xe2x80x9cshortxe2x80x9d with 60-62 residues and four disulfides, or xe2x80x9clongxe2x80x9d with 66-74 residues and a fifth disulfide. The long xcex1-neurotoxin, xcex1-bungarotoxin (xcex1Bgtx), from the venom of Bungarus multicinctus, has played a critical role in the biochemical purification and characterization of nAChRs from muscle (e.g., Changeux et al., Proc. Natl.Acad Sci. USA 67:1241-1247, 1970) and brain (Whiting and Lindstrom, Biochem. 25:2082-2093, 1986; Whiting and Lindstrom, Proc. Nati. Acad Sci. USA 84:595-599, 1987) due to the essentially irreversible binding of aBgtx to these receptors.
Venom-derived xcex1Bgtx has been a critically important tool in biochemical studies of nAChRs largely because of its high affinity and nearly irreversible binding characteristics (Chiappinelli, 1995). Similar biochemical studies of most neuronal nAChRs, apart from xcex17 subunit-containing receptors, have been hampered by the lack of similar high-affinity probes. xcexa-Bungarotoxin blocks cholinergic transmission in peripheral nervous tissue (e.g., Chiappinelli et al., Toxicon 34:1243-1256, 1996) and has a high specificity for xcex13xcex22 receptors (Grant et al., Biochem. 37:12166-12171, 1998), but this toxin is in relatively short supply. Recently, two naturally-occurring toxins isolated from the venom of Conus snails have been shown to be selective for xcex13xcex22 and xcex13xcex24 nAChRs (Cartier et al., J. Biol. Chem. 271:7522-7528, 1996; Luo et al., J. Neurosci. 18:8571-8579, 1998) although their utility may be limited by their very rapid dissociation kinetics. Thus there is a need for additional well characterized molecular probes of high specificity for investigation of the properties of nAChRs, particularly xcex17 subunit-containing nAChRs.
Bacterial, snake and snail toxins typically affect neurotransmitter uptake and/or release at neuromuscular synapses. Despite their lethality, a variety of these toxins are used as therapeutic agents in human disease. For example, treatment of certain neuromuscular disorders involving local muscle spasticity or dystonia (see, e.g., U.S. Pat. Nos. 5,721,215; 5,677,308; 5,562,907, 5,053,005, etc.) involves injection of a chemodenervating agent, currently a botulinum toxin preparation (BOTOX(copyright), Allergan, Irvine, Calif.), directly into the muscle, using, for example, a fine gauge teflon-coated needle under electromyographic control to aid the physician in locating the intended intramuscular locus of the injection. A sufficient dose of the toxin acts on striated muscle to block release of the acetylcholine neurotransmitter from the presynaptic membrane resulting in varying degrees of effective denervation of the muscle in regions contacted by the toxin (i.e., causing local paralysis). This results in an increase in post-synaptic acetylcholinesterase activity and an increase in the population of acetylcholine receptors, effects which occur as a characteristic physiological response to denervation. After a period of days, the axon terminals develop sprouting, and over a period of several months, collateral motor axons establish new neuromuscular connections with the muscle fiber. As neuromuscular junctions are regenerated, full function of the muscle returns along with the spasmodic contractions or hyperstimulation symptomatic of the disease.
Botulinum toxin treatment is also known to be associated with a number of side effects. Such side effects include transient fatigue, dysphagia, neck weakness, hoarseness and localized pain. In addition, many patients who preliminarily respond to botulinum toxin therapy subsequently become non-responsive to the treatment. Accordingly, for many patients the botulinum injections fail to provide satisfactory long term treatment of the condition. Thus there is a need to develop additional chemodenervating agents that do not have the foregoing deficiencies of botulinum toxin.
The invention provides compositions and methods for nonpermanent and localized inhibition of neuronal cell function. It has now been discovered that xcex1-bungarotoxin and modified xcex1-bungarotoxin molecules are useful for reducing neurotransmitter effects at neuromuscular junctions, thereby inducing the temporary paralysis of muscles. These effects are useful for the treatment of aberrant muscle contraction, inter alia in the cosmetic treatment of facial wrinkles, in strabismus, blepharospasm, various dystonias and other conditions having neuromuscular components.
According to one aspect of the invention, methods of enhancing relaxation or slackening of cutaneous tissue are provided. The methods include locally administering to a cutaneous tissue an isolated native or modified xcex1-bungarotoxin molecule in an amount effective to enhance denervation of the muscle or group of muscles present subcutaneous to the cutaneous tissue to enhance relaxation or slackening of the cutaneous tissue. In some embodiments, the xcex1-bungarotoxin is administered subcutaneously.
Preferably the relaxation or slackening of the cutaneous tissue results in lessening of wrinkles or fine lines of the skin. In some embodiments the methods further include co-administering an anti-wrinkle agent selected from the group consisting of hydroxy acids and retinoids. In preferred embodiments the hydroxy acid is selected from the group consisting of xcex1-hydroxy acids and xcex2-hydroxy acids, which can be linear, branched or cyclic and saturated or unsaturated and the retinoid is selected from the group consisting of retinoic acid, retinol and retinol esters.
In certain embodiments, the xcex1-bungarotoxin molecule is an isolated native xcex1-bungarotoxin molecule or a fragment thereof, preferably one that includes SEQ ID NO:2. On other embodiments, the xcex1-bungarotoxin molecule is an isolated modified xcex1-bungarotoxin molecule or a fragment thereof. Preferred modified xcex1-bungarotoxin molecules include those having at least one amino acid substitution selected from the group consisting of a substitution at amino acid 38 and a substitution at amino acid 42 of SEQ ID NO:2.
According to another aspect of the invention, methods of treating spasm or involuntary contraction in a muscle or a group of muscles in a subject are provided. The methods include administering to a muscle or a group of muscles in a subject in need of such treatment an isolated native or modified xcex1-bungarotoxin molecule, in an amount effective to inhibit spasm or involuntary contraction in the muscle or the group of muscles of the subject.
In some embodiments the subject in need of such treatment has blepharospasm, strabismus, spasmodic torticollis, focal dystonia, jaw dystonia, occupational dystonia, corneal ulceration (protective ptosis), spasmodic dysphonia (laryngeal dystonia), or facial dyskinesis such as Meige syndrome, hemifacial spasm, aberrant regeneration of facial nerves, or apraxia of eyelid opening.
In certain embodiments, the xcex1-bungarotoxin molecule is an isolated native xcex1-bungarotoxin molecule or a fragment thereof, preferably one that includes SEQ ID NO:2. On other embodiments, the xcex1-bungarotoxin molecule is an isolated modified xcex1-bungarotoxin molecule or a fragment thereof. Preferred modified xcex1-bungarotoxin molecules include those having at least one amino acid substitution selected from the group consisting of a substitution at amino acid 38 and a substitution at amino acid 42 of SEQ ID NO:2.
According to yet another aspect of the invention, methods of controlling autonomic nerve function in a subject are provided. The methods include locally administering to a target tissue or organ of a subject in need of such treatment an isolated native or modified xcex1-bungarotoxin, in an amount effective to enhance denervation in the target tissue or organ and control autonomic nerve function in the subject.
In some embodiments, the autonomic nerve function includes the function of an autonomic nerve which contributes to at least one symptom of rhinorrhea, otitis media, excessive salivation, asthma, chronic obstructive pulmonary disease, excessive stomach acid secretion, spastic colitis or excessive sweating.
In certain embodiments, the xcex1-bungarotoxin molecule is an isolated native xcex1-bungarotoxin molecule or a fragment thereof, preferably one that includes SEQ ID NO:2. On other embodiments, the xcex1-bungarotoxin molecule is an isolated modified xcex1-bungarotoxin molecule or a fragment thereof. Preferred modified xcex1-bungarotoxin molecules include those having at least one amino acid substitution selected from the group consisting of a substitution at amino acid 38 and a substitution at amino acid 42 of SEQ ID NO:2.
According to still another aspect of the invention, isolated polypeptide which selectively bind nicotinic acetylcholine receptors with a non-native specificity are provided. The polypeptides include the amino acid sequence of SEQ ID NO:2 having at least one amino acid substitution, or a fragment thereof.
In certain embodiments the at least one amino acid substitution is selected from the group consisting of a substitution at amino acid 38 and a substitution at amino acid 42 of SEQ ID NO:2. Preferably the at least one amino acid substitution is selected from the group consisting of Pro at amino acid 38 and Gln at amino acid 42 of SEQ ID NO:2. More preferably, the isolated polypeptide includes the amino acid sequence of SEQ ID NO:2 having amino acid substitutions of Pro at amino acid 38 and Gln at amino acid 42.
Pharmaceutical compositions, including the foregoing isolated polypeptides and a pharmaceutically acceptable carrier, also are provided according to the invention. Preferably a pharmaceutically effective amount of the isolated polypeptides is included in the pharmaceutical composition.
According to another aspect of the invention, pharmaceutical composition are provided that include an isolated native xcex1-bungarotoxin and a pharmaceutically acceptable carrier.
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments.